Project Summary It is vital to understand more about the dynamics of aging, their interaction with environmental factors, and the connections between disease processes and aging for the prevention and treatment of age-related diseases. Our work on environmental enrichment (EE), a housing environment boosting mental health, has revealed a novel anticancer and anti-obesity phenotype mediated by a brain-fat axis: the hypothalamic-sympathoneural- adipocyte (HSA) axis where hypothalamic BDNF is the key brain mediator. Our preliminary data show that EE promotes healthy aging in aged mice characterized as reduced adiposity, improved glucose tolerance, decreased leptin level, enhanced motor abilities, and reduced anxiety. Long-term hypothalamic overexpressing of BDNF via a novel autoregulatory AAV-BDNF vector mimics EE effects in aged mice. EE and hypothalamic BDNF overexpression also downregulate inflammatory genes in the hypothalamus of obese mice (young and old) or old mice of normal weight, including IL-1? and NF?B signaling genes which serves a key pro- inflammatory mechanism of hypothalamic microinflammation. Moreover, EE leads to significant morphological changes of hypothalamic microglia in aged mice characterized as an increase in microglia surveying area and branching without change of microglia number. Accumulating evidence suggests that both dietary excess and aging trigger accumulation of pro-inflammatory microglia in the hypothalamus, and the microglial inflammatory signaling mediates obesity susceptibility and aging-associated metabolic decline. Thus, we propose to test our hypothesis that EE regulates hypothalamic microglia via BDNF and subsequently contributing to the metabolic adaptations in both obesity and aging. We plan to investigate how EE modulates hypothalamic microglia and the role of hypothalamic microglia in EE-induced metabolic adaptations in aging and dietary excess in Aim 1. Specifically, we will assess EE effects on hypothalamic microglia phenotypes in young, middle age, and old mice (number, morphology, activation states, transcriptome, etc.). To study whether hypothalamic microglia is required for initiating or sustaining EE-induced metabolic adaptions, CSF1R kinase inhibitor PXL5622 is used to selectively deplete microglia. In order to deplete microglia specifically in the hypothalamus, microglia- targeting viral vector harboring Cre recombinase is injected to the hypothalamus of mice harboring conditional alleles of CSF1R. We will probe whether microglial NF?B mediates EE's suppression of inflammatory signaling using microglia-targeting lentiviral vector to silence IKK?, essential for NF?B activation. Moreover, we will investigate the role of hypothalamic BDNF in EE-induced microglia regulation in Aim 2. We will assess whether rAAV-mediated hypothalamic overexpressing BDNF can mimic EE's effects on microglia. Conversely, a microRNA specifically targeting Bdnf is used to study whether inhibition of hypothalamic BDNF could block EE's modulation of microglia. These studies will explore an understudied area, gain insights on how the complex process of aging interacts with lifestyle factors, and identify potential therapeutic targets.